Cellular proliferation, and differentiation of cells in response to extracellular signals, are controlled by the signal transduction pathway of Ras, Raf and MAP (mitogen-activated protein) kinase. The mechanisms that regulate this pathway are not well known. Here we describe two structurally similar tyrosine kinase substrates, Spred-1 and Spred-2. These two proteins contain a cysteine-rich domain related to Sprouty (the SPR domain) at the carboxy terminus. In Drosophila, Sprouty inhibits the signalling by receptors of fibroblast growth factor (FGF) and epidermal growth factor (EGF) by suppressing the MAP kinase pathway. Like Sprouty, Spred inhibited growth-factor-mediated activation of MAP kinase. The Ras-MAP kinase pathway is essential in the differentiation of neuronal cells and myocytes. Expression of a dominant negative form of Spred and Spred-antibody microinjection revealed that endogenous Spred regulates differentiation in these types of cells. Spred constitutively associated with Ras but did not prevent activation of Ras or membrane translocation of Raf. Instead, Spred inhibited the activation of MAP kinase by suppressing phosphorylation and activation of Raf. Spred may represent a class of proteins that modulate Ras-Raf interaction and MAP kinase signalling.

Myc is a ubiquitous mediator of cell proliferation and can transactivate the expression of various genes through E-box sites. Here we report a novel gene, mina53 (Myc-induced nuclear antigen with a molecular mass of 53 kDa). The mina53 gene encodes a protein with a molecular weight of 53 kDa, which is localized in the nucleus and with part of the protein concentrated in the nucleolus. When serum-starved cells were activated by serum, the level of c-myc mRNA was elevated, and an increase in mina53 mRNA followed the elevation of cmyc mRNA. When expression of c-myc was reduced in human promyelocytic leukemia HL60 cells by phorbol 12-myristate 13-acetate, the expression of mina53 mRNA and protein was reduced. The expression of mina53 mRNA and

The rate-limiting step in ribosome biogenesis is the transcription of ribosomal RNA, which is controlled by environmental conditions. The JmjC enzyme KDM2A/ JHDM1A/FbxL11 demethylates mono-and dimethylated Lys 36 of histone H3, but its function is unclear. Here, we show that KDM2A represses the transcription of ribosomal RNA. KDM2A was localized in nucleoli and bound to the ribosomal RNA gene promoter. Overexpression of KDM2A repressed the transcription of ribosomal RNA in a demethylase activity-dependent manner. When ribosomal RNA transcription was reduced under starvation, a cellpermeable succinate that inhibited the demethylase activity of KDM2A prevented the reduction of ribosomal RNA transcription. Starvation reduced the levels of mono-and dimethylated Lys 36 of histone H3 marks on the rDNA promoter, and treatment with the cell-permeable succinate suppressed the reduction of the marks during starvation. The knockdown of KDM2A increased mono-and dimethylated Lys 36 of histone H3 marks, and suppressed the reduction of ribosomal RNA transcription under starvation. These results show a novel mechanism by which KDM2A activity is stimulated by starvation to reduce ribosomal RNA transcription.

The hormone glucose-dependent insulinotropic polypeptide (GIP) potently stimulates insulin secretion and promotes ␤-cell proliferation and cell survival. In the present study we identified Forkhead (

Ectodomain shedding is an important mechanism to regulate the biological activities of membrane proteins. We focus here on the signaling mechanism of the ectodomain shedding of heparin-binding epidermal growth factor (EGF)-like growth factor (pro HB-EGF). Lysophosphatidic acid (LPA), a ligand for seven-transmembrane G protein-coupled receptors, stimulates the shedding of pro HB-EGF, which constitutes a G proteincoupled receptor-mediated transactivation of the EGF receptor. Experiments using a series of inhibitors and overexpression of mutant forms of signaling molecules revealed that the Ras-Raf-MEK signal is essential for the LPA-induced shedding. In addition, the small GTPase Rac is involved in the LPA-induced shedding, possibly to promote MEK activation. 12-O-Tetradecanoylphorbol-13-acetate is another potent inducer of pro HB-EGF shedding. We also demonstrate that the LPA-induced pathway is distinct from the 12-O-tetradecanoylphorbol-13-acetate-induced pathway and that these pathways constitute a dual signaling cascade that regulates the shedding of pro HB-EGF.

Mina53 is a novel Myc target gene that we previously demonstrated to be involved in cell proliferation. We studied, here, the expression of Mina53 in colon cancer to examine its possible role in carcinogenesis. We generated a specific monoclonal anti-human Mina53 antibody and found that colon tumor cell lines expressed Mina53 highly. We also found that expression

Abstract. Diphtheria toxin (DT) receptor associateswith a 27-kD membrane protein (DRAP27) in monkey Vero cells. A cDNA encoding DRAP27 was isolated, and its nucleotide sequence was determined. The deduced amino acid sequence revealed that DRAP27 is the monkey homologue of human CD9 antigen. DRAP27 is recognized by CD9 antibodies. A humanmouse hybrid cell line (3279-10) possessing human chromosome 5, sensitive to DT, but not expressing CD9 antigen, was used for transfection experiments with DRAP27. When the cloned cDNA encoding DRAP27 was transiently expressed in 3279-10 cells, the total DT binding capacity was three to four times higher than that of untransfected controls. Transfectants stably expressing DRAP27 have an increased number of DT binding sites on the cell surface. Furthermore, the transfectants are 3-25 times more sensitive to DT than untransfected cells, and the sensitivity of these cells to DT is correlated with the number of DRAP27 molecules on the surface. However, when the cloned cDNA was introduced into mouse cell lines that do not express DT receptors, neither an increased DT binding nor enhancement of DT sensitivity was observed. Hence, we conclude that DRAP27 itself does not bind DT, but serves to increase DT binding and consequently enhances DT sensitivity of cells that have DT receptors. 12 proteins related to DRAP27/ CD9 antigen were found through homology search analysis. These proteins appear to belong to a new family of transmembrane proteins.IPHTHERIA toxin (DT), ~ secreted by Corynebacterium diphtheriae, is a cytotoxic protein (Mr = 58,342) that inhibits cellular protein synthesis in eukaryotes by inactivating elongation factor 2 through ADP ribosylation (for reviews, see references 18 and 48). Entry of the toxin, or at least the A fragment, into the cytoplasm is required for the cytotoxic action to occur (65). The toxin binds to a specific receptor on the cell surface (15, 37) and is then internalized by receptor-mediated endocytosis (42,45). A conformational change of the toxin molecule takes place in an acidic compartment, resulting in the interaction of hydrophobic domains of the toxin molecule with the lipid bilayer of the compartment (10, 11, 14, 52). Finally, the enzymatically active A fragment is translocated to the cytosol (43, 44), where it inactivates elongation factor 2. The sensitivity of cells to DT varies among species. Cells from many mammals, including humans and monkeys, are sensitive to 1. Abbreviations used in this paper: DRAP27, 27-kD diphtheria toxin receptor-associated protein; DT, diphtheria toxin; RDS, retinal degeneration slow proteins.DT, but those from rats and mice are not (40). The difference in DT sensitivity among cells is primarily determined by the number of DT-specific receptors on the surface (31,36,41).The Vero cell line, derived from monkey kidney, is one of the cell lines most sensitive to DT (40). The use of Vero cells has facilitated biochemical studies of the DT receptor. Chemical cross-linking of the protein(s) on the cell surface with DT fo...

c Environmental conditions control rRNA transcription. Previously, we found that serum and glucose deprivation induces KDM2A-mediated H3K36me2 demethylation in the rRNA gene (rDNA) promoter and reduces rRNA transcription in the human breast cancer cell line MCF-7. However, the molecular mechanism and biological significance are still unclear. In the present study, we found that glucose starvation alone induced the KDM2A-dependent reduction of rRNA transcription. The treatment of cells with 2-deoxy-D-glucose, an inhibitor of glycolysis, reduced rRNA transcription and H3K36me2 in the rDNA promoter, both of which were completely dependent on KDM2A in low concentrations of 2-deoxy-D-glucose, that is, mild starvation conditions. The mild starvation induced these KDM2A activities through AMP-activated kinase (AMPK) but did not affect another AMPK effector of rRNA transcription, TIF-IA. In the triple-negative breast cancer cell line MDA-MB-231, the mild starvation also reduced rRNA transcription in a KDM2A-dependent manner. We detected KDM2A in breast cancer tissues irrespective of their estrogen receptor, progesterone receptor, and HER2 status, including triple-negative cancer tissues. In both MCF-7 and MDA-MB-231 cells, mild starvation reduced cell proliferation, and KDM2A knockdown suppressed the reduction of cell proliferation. These results suggest that under mild glucose starvation AMPK induces KDM2A-dependent reduction of rRNA transcription to control cell proliferation. R egulation of cell growth ultimately depends on the control of new ribosome synthesis, and the rate of ribosome synthesis is tightly regulated in mammalian cells (1). Three of the four structured ribosomal RNA (rRNA) molecules constituting a ribosome are produced by processing a precursor transcript, pre-rRNA. The pre-rRNA is coded by rRNA genes (rDNA) and specifically transcribed by RNA polymerase I (Pol I) in the nucleolus (1-5). Ribosome biogenesis is limited by rRNA transcription, and the control of rRNA transcription is thought to play a central role in the regulation of ribosome biogenesis and cell growth (6-9). The rRNA transcription is dysregulated during tumorigenesis, and selective inhibition of rRNA transcription may offer a therapeutic strategy to block cancer cell proliferation (3, 10, 11).The level of rRNA transcription is controlled by environmental conditions (11). To date, increasing numbers of studies have revealed that the signal transduction pathways reach the rRNA transcription machinery in the nucleolus and regulate rRNA transcription (7,9,(12)(13)(14). Some signal pathways control the activities of basic transcription factors for Pol I (2). For example, mitogen-activated protein kinase (MAPK) signaling, the mammalian target of rapamycin signaling, and type 1 insulin-like growth factor activate rRNA transcription through upstream binding factor, selectivity factor 1 and/or transcription initiation factor IA (TIF-IA, the mammalian homolog of yeast Rrn3) (15-21). AMP-activated kinase (AMPK), a sensor to maintain energy homeo...